Detection technologies for RNA modifications

To date, more than 170 chemical modifications have been characterized in RNA, providing a new layer of gene expression regulation termed the ‘epitranscriptome’. RNA modification detection methods and tools advance the functional studies of the epitranscriptome. According to the detection throughput and principles, existing RNA modification detection technologies can be categorized into four classes, including quantification methods, locus-specific detection methods, next-generation sequencing-based detection technologies and nanopore direct RNA sequencing-based technologies. In this review, we summarize the current knowledge about these RNA modification detection technologies and discuss the challenges for the existing detection tools, providing information for a comprehensive understanding of the epitranscriptome.

INTRODUCTION
RNA is a single-stranded molecule consisting of four nucleotides: adenosine (A), guanosine (G), cytidine (C) and uridine (U). RNA, both an important and conserved macromolecule, not only participates in the flow of genetic information but also regulates gene expression. Beyond sequence information, chemical modifications add to the complexity of RNA, emerging as a new layer of gene expression regulation. Since the first chemical modification was characterized 60 years ago, more than 170 RNA modifications have been characterized 1 . Most of the RNA modifications have been identified in abundant non-coding RNAs, including ribosomal RNA (rRNA), transfer RNA (tRNA), and small nuclear RNA (snRNA).
RNA modification detection technologies provide not only resources for a comprehensive understanding of the epitranscriptome but also tools for functional studies. Hence, in this review, we will summarize the current knowledge about these existing RNA modification detection technologies and discuss the challenges for these existing detection tools. The detection technologies are categorized according to the detection throughput and principles into four classes: quantification methods, locus-specific detection methods, next-generation sequencing-based detection technologies and nanopore direct RNA sequencing-based technologies.

RNA MODIFICATION QUANTIFICATION METHODS
The identification and quantification of new modified nucleotides requires powerful RNA modification quantification methods. Based on the principle that modified nucleotides possess distinct chemical properties from the originals, several RNA modification quantification methods have been established, including twodimensional thin-layer chromatography (2D-TLC), dot blot, and liquid chromatography-mass spectrometry (LC-MS). These approaches can be used to quantify the modification abundance in specific RNA species and require highly purified RNA due to the lack of sequence information.
2D-TLC 2D-TLC is a widely used RNA modification detection method according to the distinct mobilities of different nucleotides in the solvent [60][61][62] . In detail, isolated RNA is first partially digested into oligonucleotides using RNase A, T1, or T2 and then labeled with 32 P using T4 polynucleotide kinase (T4 PNK). Finally, 5ʹ-32 P-NMP is acquired by nuclease P1 digestion and further separated by 2D-TLC. The nucleotides can be determined by assignment to the standards by comparing their retardation factor (Rf) values (Fig. 1). Quantification of the nucleotides is achieved by measuring the radioactivity of the corresponding spots in the TLC plate [63][64][65] . This approach is very sensitive and requires only a small amount of RNA (from 50 ng to 200 ng). Consequently, this approach can be applied to both abundant non-coding RNAs (rRNA and tRNA) and less abundant mRNA. In addition, it can also detect RNA modifications in specific tRNA or snRNA sequences, which can be isolated by gel purification or hybridization methods. Furthermore, this approach does not require expensive instruments and thus can be run inexpensively. However, this method also has certain drawbacks, including the requirement of a radioactive reagent, the bias caused by RNase digestion and discrepant 32 P labeling efficiency for the modified nucleotides 63,66 .

Dot blot
Dot blot assay is established and applied to detect and quantify modification levels in RNAs using a specific antibody. In detail, isolated RNAs are stalled to the polyvinylidene fluoride (PVDF) or nitrocellulose membrane directly without electrophoretic size separation. A specific antibody for target modification is incubated with the membrane, followed by secondary antibody hybridization and subsequent signal trapping (Fig. 1). Comparing results from different experimental groups can obtain semiquantitative modification level information. This approach has been widely applied to various RNA species, including noncoding RNAs and mRNAs. Given the detection principle, the sensitivity and accuracy of this approach are highly dependent on the specificity of the antibody. In addition, the amount of starting material, ranging from nanograms to micrograms, also depends on the abundance of the RNA modifications of interest. Since the workflow is straightforward and can be performed inexpensively, this approach has been widely applied for RNA modification detection 67,68 . However, the lack of absolute quantification and locus information limits a wider application of this approach.

HPLC and LC-MS
High-performance liquid chromatography (HPLC) is an advanced column chromatography technology to separate nucleosides according to their distinct polarities. Prior to HPLC analysis, RNA or oligonucleotides are digested and dephosphorylated to single nucleosides by nuclease P1 and alkaline phosphatase. The UV absorbance and retention time of the nucleosides are recorded by a UV detector, which can be used to identify and measure the abundance of the modified nucleosides ( Fig. 1). Compared with 2D-TLC, HPLC analysis is rapid and free of radiolabeling. However, this approach can only be applied to detect highly abundant modifications in abundant RNA species, such as rRNA, tRNA and synthetic RNAs, and requires a large amount of purified RNA (more than 1 µg) due to the detection limit of the UV detector 69 .
To increase detection sensitivity, HPLC is coupled with mass spectrometry 70 . Similarly, RNA or oligonucleotides are completely digested to nucleosides and separated by reverse column chromatography. Then, these nucleosides are ionized and further fragmented into specific product ions via mass spectrometry. Integration of retention time, mass-to-charge ratio (m/z) and product ion are capable of determining a certain nucleoside (Fig.  1). In addition, quantification of nucleosides can be achieved through the external standard curve in the same batch 60,71 . The extremely high sensitivity of triple quadrupole-based mass spectrometry provides this approach a detection limit that can reach the low femtomolar range, and the amount of starting material can be as low as 50 ng, thereby allowing the determination and quantification of these low-abundance modifications in mRNA and low-abundance ncRNA 72,73 . Hence, LC-MS has been a benchmark for RNA modification detection and quantification. However, the limit of this technique is the requirement of instruments including HPLC and mass spectrometry. In addition, due to the lack of sequence information, when detecting and quantifying RNA modifications in mRNA or other less abundant RNA types, caution must be taken to reduce contamination from abundant and highly modified rRNA and tRNA.

LOCUS-SPECIFIC DETECTION METHODS
The precise position information of a certain RNA modification is important for functional studies. To date, several locus-specific RNA modification detection methods have been developed. These methods can be categorized according to the detection principles into four classes: (1) Primer extension; (2) RNase H-based approaches; (3) electrospray ionization-mass spectrometry (ESI-MS)-based approaches; and (4) semiquantitative PCR-or qPCRbased approaches.

Primer extension
This approach is based on reverse transcription and has been used extensively to detect and localize various RNA modifications, including m 1 A, Ψ, and m 1 G. In principle, a 5ʹ-labeled specific RT primer is hybridized with the RNA of interest and extended by reverse transcriptase. Hence, this approach relies on prior knowledge of the modification type and sequence information of the target RNA. Without modifications, reverse transcriptase can reach the 5ʹ end of RNA and generate full-length cDNA. When encountering modified nucleotides, the extension of reverse transcriptase is blocked immediately upstream of the modified site. Then, the RT products are separated using denaturing polyacrylamide gels, and the terminal position of truncated cDNA indicates where the modification occurred (Fig. 2a). This approach is very sensitive and can be applied to various RNA species, including rRNA, tRNA, snRNA and abundant mRNA 42,43,[74][75][76][77][78][79][80] . In addition, owing to the hybridization step, this approach possesses high detection specificity and does not require purified homogenous RNA as the starting material. A major limitation of this method is that it can only detect the modifications or their chemical adducts that are capable of blocking reverse transcription; it is not suitable for RT-silent modifications such as m 6 A and m 5 C 81-83 .

RNase H-based approach
The RNase H-based approach is independent of reverse transcription and thus can detect and quantify RNA modifications that do not affect Watson-Crick base pairing. In this method, purified RNA is cleaved into two halves at the 5ʹ end of the nucleotide of interest by RNase H, and cleavage specificity is achieved by annealing with a specific 2′-O-methyl RNA-DNA chimera oligonucleotide. Then, the 3ʹ-half of the RNA is purified, and its 5′ terminus is further labeled with 32 P. Furthermore, the oligonucleotides are completely digested into single nucleotides and subsequently resolved by TLC. Owing to the radiolabeling specificity, only the modified and unmodified forms of the nucleotide of interest can be detected and accurately quantified by TLC analysis 64,84 . This approach is sensitive and powerful for quantifying the stoichiometry of modified nucleotides. However, as the specific 2′-O-methyl RNA-DNA chimera oligonucleotide is base-paired with the target RNA sequence to guide RNase H cleavage, sequence information is needed, and modifications that affect Watson-Crick base pairing cannot be detected and quantified by this method. In addition, to reduce false signals from other RNA sequences, homogenously purified RNA, isolated by the hybridization method and gel purification, is used as the starting material. Therefore, this method requires a large amount of RNA and can be applied to abundant non-coding RNAs, such as rRNA and snRNA 64,84 .
To quantitatively detect modifications in mRNA/lncRNA, a new method, named SCARLET (site-specific cleavage and radioactive labeling followed by ligation-assisted extraction and thin-layer chromatography), has been developed 85 . In SCARLET, a 116nucleotide single-stranded DNA oligonucleotide is splint-ligated to the 32 P-labeled RNA fragment of interest and then subjected to RNase T1/A digestion. The splint ligation enables RNA mixtures to be used as the starting material rather than a specific purified RNA, and modifications in mRNA and lncRNA can thus also be quantified. The accurate size of the DNA oligonucleotide is recovered by gel purification. Then, the 32 P-labeled nucleotides are released by nuclease P1 digestion and further analyzed by TLC (Fig. 2b). This approach has been applied to quantify m 6 A and Ψ modification status in mRNA 85,86 . Since this approach avoids reverse transcription, it can be used as an orthogonal method to validate modification sites identified by transcriptome-wide methods that are dependent on reverse transcription. As with other RNase H-based methods, SCARLET also requires prior knowledge of the sequence information to target a single specific nucleotide and cannot quantify the modification status in a de novo manner. In addition, the requirement of radioactive reagents and complicated procedures limit wider application of this method.
Semiquantitative PCR or qPCR-based approach Similar to primer extension assays, semiquantitative PCR or qPCRbased approaches are also based on the fact that modified nucleotides impede reverse transcriptase extension. In contrast to primer extension and RNase H-based approaches, qPCR-based approaches are free of radiolabeling and hence are time-saving and easy to perform in the laboratory. To date, several semiquantitative PCR-or qPCR-based approaches have been developed and have successfully detected Nm, Ψ and m 6 A in diverse RNA species (Fig. 2c-e) [87][88][89][90][91][92][93] . In addition, owing to the high sensitivity and specificity of PCR and qPCR, these approaches do not require purified RNA as the starting material and can be applied to various RNA species, including less abundant mRNA and lncRNA.
Nm blocks reverse transcriptase extension at a low dNTP concentration and allows readthrough at a high dNTP concentration 82,94 . Taking advantage of this property, researchers have developed a method referred to as RTL-P (reverse transcription at low dNTP concentrations followed by PCR). In this method, the RNA of interest is first reverse-transcribed at both low and high dNTP concentrations. The truncated and full-length cDNA are amplified by specific primers. The PCR products are analyzed by gel electrophoresis, and the Nm status can be determined by comparing the intensity of the longer PCR product with that of the shorter product ( Fig. 2c) 87 . In addition to altering RT conditions, RT enzymes can also be engineered to facilitate modified nucleotide detection. For instance, an engineered thermostable KlenTaq DNA polymerase variant possesses reverse transcription activity and can discriminate Nm at normal dNTP concentrations. The combination of this engineered DNA polymerase with qPCR has achieved expeditious quantification of Nm 88 .
In addition to the engineered thermostable KlenTaq DNA polymerase variant, two other DNA polymerases, Tth and Bst, also have reverse transcriptase activity and exhibit distinct capacities to extend when encountering m 6 A residues versus A residues, which allows locus-specific detection of m 6 A 89-91 . Moreover, to increase the detection sensitivity of m 6 A, another method referred to as the single-base elongation-and ligation-based PCR amplification method (SELECT) has been developed 92,93 . Because m 6 A can both hinder the elongation activity of DNA polymerases and reduce the nick ligation efficiency of SplintR ligase, in SELECT, cDNA products formed from m 6 A-containing RNA templates are dramatically reduced, hence significantly increasing detection robustness. Based on qPCR, SELECT is a powerful tool to quantify the m 6 A fraction in a linear manner (Fig. 2d).
In addition to truncations, the property of induced mutation/ deletions by modified nucleotides can also be utilized for locusspecific detection. For instance, Ψ can be selectively labeled by Ncyclohexyl-N′-(2-morpholinoethyl) carbodiimide (CMC), and the Ψ-CMC adducts interfere with Watson-Crick base pairing during reverse transcription. Under optimized RT conditions, Ψ-CMC adducts can be read through and induce mutation/deletion in cDNA. Such mutation/deletions alter the melting curves of the qPCR products, thus enabling the locus-specific detection of Ψ modification (Fig. 2e) 95 .

ESI-MS-based approach
In contrast to the LC-MS-based nucleoside quantification strategy described above, in this approach, isolated RNA samples are first digested into 5-15 nucleotide fragments by selective endoribonucleases, such as RNase T1, RNase A, and RNase U2. Then, the oligonucleotides are separated by HPLC, and the sequence ladders from the oligonucleotides are further generated through ESI-MS, which can be used for sequence reconstruction and modification identification (Fig. 2f). Hence, this approach can provide both site and stoichiometry information for the modifications of interest 70,[96][97][98][99] . Compared with other locus-specific methods, the ESI-MS-based approach does not rely on prior knowledge of sequence information and thus is able to detect and quantify RNA modification in a de novo manner. Therefore, this approach has been widely applied for modification detection in abundant RNAs, including tRNA, rRNA and snRNA [100][101][102][103][104][105][106][107][108][109] . Recently, this approach has been used to detect modifications in miRNA and cap modifications in mRNA 53,110 . The major limitation of this approach is the requirement for highly sensitive ESI-MS. In addition, given the detection principle, the requirement of starting material is large; thus, this method is only suitable for abundant RNA species.

NEXT-GENERATION SEQUENCING-BASED DETECTION TECHNOLOGIES
With the benefit of advances in next-generation sequencing, an increasing number of RNA modification sequencing technologies have been developed. Such technologies represent powerful tools to map modified nucleotides in a transcriptome-wide manner and promote the elucidation of the regulatory roles of RNA modifications. Chemical modifications alter the inherent features of the original nucleotides, including base-pairing performance in reverse transcription, chemical reaction activities, enzymatic reaction activities and binding affinities with specific proteins or antibodies. Therefore, coupled with next-generation sequencing, these properties of modified nucleotides can be used to characterize RNA modifications throughout the transcriptome. The existing sequencing technologies can be categorized according to the detection principles into four classes: (1) direct sequencing technology, (2) chemical-assisted sequencing technology, (3) antibody-based sequencing technology, and (4) enzyme/protein-assisted sequencing technology (Table 1).

Direct sequencing technology
For some modified nucleotides, the existence of chemical modifications alters canonical Watson-Crick base pairing during reverse transcription and further leads to truncation or misincorporation in cDNA synthesis. Therefore, this feature can be used to map modified nucleotides throughout the transcriptome.
For instance, in contrast to adenosine, inosine pairs with cytidine in reverse transcription, and thus, the A-to-I editing position can be identified by comparing the genomic DNA and RNA sequencing data and detecting A-to-G mismatch sites (Fig.  3a) [111][112][113][114] . Although this approach is widely applied to A-to-I editing site detection, caution is still needed to reduce the false positives introduced by SNPs, somatic mutations, pseudogenes and sequencing errors 22 .
Aside from inosine, several other chemical modifications at the Watson-Crick face of the nucleobase can also induce RT stops or mutations in cDNA synthesis, thereby allowing base-resolution detection 115 . In contrast to inosine, the reverse transcription signatures of these modifications are not constant and dependent on both the surrounding sequences and the RT conditions 116 . Furthermore, to increase the detection accuracy, several improvements have been made. For instance, the E. coli AlkB demethylase and its mutant treatment are introduced to remove m 1 A, m 3 C, m 1 G, N 2 ,N 2 -dimethyl-guanosine (m 2 2 G), and 3-methyluridine (m 3 U) in RNA prior to cDNA synthesis, and high-confidence methylation sites in the tRNA transcriptome can thus be identified by comparing the data for parallel sequenced, demethylasetreated and untreated samples (Fig. 3b) [117][118][119][120] . Moreover, the RT signatures vary substantially under different reverse transcription conditions 42,121 . Hence, to improve detection sensitivity, an HIV-1 reverse transcriptase against m 1 A was developed that allows m 1 A detection in human mRNA 122 . Moreover, RT systems, including enzymes and reaction conditions, can be optimized to detect modified nucleotides that do not interfere with Watson-Crick base pairing. For instance, Nm, an RT-silent modification, can block cDNA synthesis when the concentration of dNTPs or Mg 2+ in RT reactions is limited. Coupling this feature with next-generation sequencing, two methods, 2OMe-seq and MeTH-seq, have achieved a transcriptomic profile of Nm at base resolution ( Fig.  3b) 123,124 . As mentioned above, some DNA polymerases also possess reverse transcriptase activity and show differential elongation ability when encountering m 6 A residues versus A residues. To advance this feature, the KlenTaq DNA polymerase was evolved and the evolved variant exhibited significantly increased error rates opposite m 6 A but not unmodified A, enabling direct identification of m 6 A by analyzing the mutational signal from sequencing data (Fig. 3b) 125 . In addition, substitution of 4SedTTP (atom-specific replacement of oxygen with selenium at the 4-position) for dTTP under RT conditions can also facilitate m 6 A detection. In principle, compared with 4SeT-A, 4SeT-m 6 A pairing is unfavorable and results in aborted cDNA synthesis opposite m 6 A sites, thereby allowing for the discrimination of m 6 A from A. Furthermore, with the assistance of the m 6 A demethylase FTO through high-throughput sequencing, m 6 A can be precisely identified within the mammalian transcriptome at singlenucleotide resolution (Fig. 3b) 126 .

Chemical-assisted sequencing technologies
Chemical treatments are widely exploited to discriminate modified nucleotides from unmodified nucleotides in three ways: (1) installing biotin tags to enrich modified transcripts; (2) altering the base-pairing features to induce misincorporation or truncation in reverse transcription; and (3) chemical-induced cleavage followed by specific adaptor ligation (Fig. 4).
The enrichment strategy facilities RNA modification detection in low-abundance RNA species and low-stoichiometry modifications. Some modified nucleotides can be labeled by specific chemical reactions and further conjugated with the biotin molecules, thus enabling streptavidin enrichment. For instance, in borohydride reduction sequencing (BoRed-seq), total RNA is treated with NaBH 4 and subsequently exposed to low pH to generate abasic sites at m 7 G positions, which can be tagged with biotin molecules for further streptavidin pulldown (Fig. 4a) 53 . Direct m 6 A labeling is Fig. 2 Schemes of locus-specific RNA modification detection methods. a Technical flowchart of primer extension. The truncated cDNA product indicates where the modification occurs. b Technical flowchart of SCARLET, an RNase H-based approach; the modified and unmodified form of the nucleotide of interest can be accurately quantified by TLC; TAP, thermosensitive alkaline phosphatase. c Technical flowchart of RTL-P, a semiquantitative PCR-based approach; Nm impedes RT under low-dNTP conditions and allows readthrough under high-dNTP conditions, and the Nm status can thus be determined by comparing the intensity of the longer PCR product with that of the shorter product. d Technical flowchart of the SELECT, qPCR-based approach; this approach utilizes the feature that m 6 A hinders the elongation of Bst and reduces the ligation efficiency of SplintR to distinguish modified and unmodified RNA. e Technical flowchart of the qPCR-based Ψ detection approach; this approach utilizes the feature that the Ψ-CMC adducts induce mutation/deletion in cDNA, which alters the melting curves of the qPCR products. f Technical flowchart of the ESI-MS-based approach. Modifications that can be detected by the corresponding approaches are listed below. difficult due to the inert reactivity of the methyl group. To solve this challenge, researchers developed a method named m 6 A-SEAL-Seq (an FTO-assisted m 6 A selective chemical labeling method). In this method, m 6 A is first oxidized to hm 6 A by the demethylase FTO, and hm 6 A is further converted to N 6dithiolsitolmethyladenosine (dm 6 A) through DTT-mediated thiol addition. The free sulfhydryl group of dm 6 A enables biotin to be conjugated to the m 6 A-modified transcripts, thereby facilitating streptavidin enrichment and sequencing (Fig. 4a) 127 .
The detection strategy of altering the base-pairing properties of nucleotides by chemical treatment has been widely applied to the transcriptome-wide detection of various modifications, including inosine, m 5 C, Ψ, m 7 G, ac 4 C, D and m 6 A. For inosine detection, the direct sequencing approach is disturbed by background noise. To overcome this limitation, a selective chemical labeling reaction with acrylonitrile is adopted, and the formed N 1 -cyanoethylinosine (ce 1 I) blocks reverse transcription, resulting in truncation of cDNA. Coupling this chemical reaction with sequencing, referred to as inosine chemical erasing sequencing (ICE-seq), can achieve base-resolution inosine detection throughout the transcriptome (Fig. 4a) 128 . Bisulfite treatment selectively converts unmethylated cytidine into uridine, leading to a C-to-T transition in the sequencing data; thus, transcriptome-wide m 5 C can be characterized by detecting nonconverted Cs in the sequencing data ( Fig. 4a) 129,130 . Furthermore, to reduce false positives caused by insufficient conversion, several improvements have been exploited, including optimizing bisulfite treatment conditions to increase deamination efficiency 131,132 , using ACT random hexamers devoid of Gs to avoid copying inefficiently deaminated RNA templates 16 , and developing robust computational pipelines to accurately identify m 5 C sites 132,133 . Ψ can be labeled by CMC at the Watson-Crick face, and the CMC-Ψ adducts stall reverse transcription, thus inducing truncations in cDNA synthesis. Combining this chemical reaction with next-generation sequencing, researchers developed Ψ-Seq, Pseudo-seq and PSI-seq, achieving base-resolution pseudouridylation detection in yeast and mammalian transcriptomes [134][135][136] . To improve the robustness of Ψ detection, the chemical reaction is adapted by using a synthesized CMC derivative, azido-CMC (N 3 -CMC), instead of CMC. The presence of an azido group enables conjugation with biotin through a click reaction. Hence, the Ψ-containing RNA can be pre-enriched before sequencing, and this method is named CeU-seq 86 . The pre-enrichment step enables CeU-seq to identify thousands of Ψ sites in the mammalian transcriptome (Fig. 4a). In addition to CMC labeling, a recent work showed that Ψ can form a stable monobisulfite adduct upon bisulfite treatment and further leave a deletion signature at the exact modified sites, thereby providing an orthogonal Ψ detection strategy (Fig. 4a) 131 . As described above, m 7 G residues can be converted to abasic sites upon NaBH 4 treatment and further recorded as misincorporations through reverse transcription and sequencing. Accordingly, the m 7 G Mutational Profiling sequencing (m 7 G-MaP-seq) can map internal m 7 G modifications at nucleotide resolution in tRNA and rRNA 137 . To achieve m 7 G detection in mRNA, m 7 G-seq conjugates a biotin molecule to the generated abasic sites, thus enabling pre-enrichment of m 7 G-containing RNA. Moreover, the biotinylated sites induce misincorporations during cDNA synthesis; therefore, transcriptome-wide base-resolution m 7 G mapping can be achieved (Fig. 4a) 54 . In addition, D can also be reduced by NaBH 4 treatment, and the reduction product can be further labeled by Rho (rhodamine). Furthermore, the Rho-adducts block reverse transcriptase elongation and thus can be identified by analyzing induced RT-stops in sequencing data (Fig. 4a) 138 . ac 4 C can react with NaCNBH 3 under acidic conditions, and the formed reduced nucleobase, N 4 -acetyltetrahydrocytidine, causes misincorporation during cDNA  166,167 synthesis. Taking advantage of this reaction, ac 4 C-seq can map ac 4 C at single-nucleotide resolution (Fig. 4a) 139 . In addition to altering the structure of nucleotides by chemical reactions, the chemical group can also be introduced through metabolic labeling. For instance, when feeding cells with an S-adenosyl methionine (SAM) analog, Se-allyl-L-selenohomocysteine, the cellular RNAs could be modified with N 6 -allyladenosine (a 6 A) at supposed m 6 A-generating sites. Furthermore, a 6 A-containing RNAs can be enriched by a specific antibody, and a 6 A sites are converted to N 1 ,N 6 -cyclized adenosine (cyc-A) through the iodination-induced cyclization reaction. As cyc-A induces misincorporations in cDNA synthesis, m 6 A can be mapped at base resolution by detecting mutation signals in sequencing data (Fig.  4b) 140 . With the exception of metabolic labeling, the allyl group can also be transferred to m 6 A by the Dim1/KsgA family of dimethyltransferases, which can specifically convert m 6 A into allylmodified m 6 A (N 6 -allyl, N 6 -methyladenosine, a 6 m 6 A). Since cyclized a 6 m 6 A can induce misincorporation in RT, the technology, named m 6 A-SAC-seq, achieves quantitative, transcriptome-wide mapping of m 6 A at single nucleotide resolution 141 . Compared with unmodified nucleotides, modified nucleotides exhibit distinct resistance under chemical hydrolysis treatment. Based on this principle, several sequencing methods have been developed and have achieved transcriptome-wide profiling for Nm, m 7 G, m 3 C and Ψ. Nm is resistant to alkaline hydrolysis and thus can be mapped at single-base resolution by analyzing read-ends information in sequencing data. Since RNA fragmentation is random and irregular, this strategy requires rather high read coverage and is limited to highly abundant RNAs [142][143][144] . To overcome this limitation, ribose oxidation sequencing (RibOxi-seq) and Nm-seq have been developed. In these two methods, RNA is treated with iterative oxidation-elimination-dephosphorylation (OED) cycles to remove unmodified nucleotides, and the nonmethylated ends cannot be ligated to linkers for sequencing library construction 145,146 . The selective cleavage and ligation allow detection of low-stoichiometry 2ʹ-O-methylation sites using this approach. Similarly, m 7 G and m 3 C are resistant to NaBH 4aniline treatment, and the generated 5′-phosphate end during aniline cleavage could be exploited for selective ligation to enrich modified fragments 147,148 . This positive selection strategy facilitates transcriptome-wide detection of m 7 G and m 3 C. In addition, upon hydrazine-aniline treatment, RNA can be specifically cleaved at m 3 C-modified sites and resistant to cleavage at Ψ-modified sites. Based on this approach, hydrazine-aniline cleavage sequencing (HAC-seq) and HydraPsiSeq have been developed to detect m 3 C and Ψ, respectively (Fig. 4a) 149,150 .

Antibody-based sequencing technologies
Antibody-based strategies have been exploited for transcriptomewide mapping of several RNA modifications, including m 6 A/ m 6 Am, m 1 A, hm 5 C, ac 4 C and m 7 G (Fig. 5) 39,[53][54][55]148,[151][152][153][154] . In this strategy, isolated RNA is first fragmented to 100-200 nt, and  detection for m 6 A and m 7 G, respectively (Fig. 5a) [155][156][157] . In addition, coupling antibody immunoprecipitation with enzyme/ chemical treatment can not only increase detection resolution but also reduce false positives. For example, taking advantage of the fact that the m 1 A-induced mutational RT signatures can be erased by AlkB demethylase treatment or Dimroth rearrangement, m 1 A-MAP, m 1 A-seq and m 1 A-IP-Seq have achieved base-resolution m 1 A methylome detection (Fig. 5a) 42,122,158 . In addition, utilizing selective in vitro demethylation for m 6 Am, m 6 Am-seq has the capability of discriminating m 6 Am from m 6 A and can identify m 6 Am at base resolution (Fig. 5b) 159 .

ENZYME/PROTEIN-ASSISTED SEQUENCING TECHNOLOGIES
In addition to antibody immunoprecipitation, some enzymes or RNA modification-related proteins can also be utilized for affinity capture or editing modification-containing transcripts, thereby enabling transcriptomic RNA modification detection (Fig. 6). For instance, by feeding a 5-aza-C analog or overexpressing mutated Nsun2, a covalent bond can be formed between m 5 C methyltransferase and its target sites. Based on this approach, Aza-IP (5azacytidine-mediated RNA immunoprecipitation) and miCLIP (methylation iCLIP) can enrich target sites by immunoprecipitation, thereby enabling identification of the direct targets of m 5 C methyltransferases (Fig. 6a) 160,161 . In addition to methyltransferase, reader proteins can also be used to target modified nucleotides. For example, in DART-seq (deamination adjacent to RNA modification targets), the cytidine deaminase APOBEC1 is fused to the m 6 A-binding YTH domain and thus leads to C-to-U deamination at sites adjacent to m 6 A residues. Furthermore, m 6 A residues can be identified by analyzing C-to-T mismatches in sequencing data (Fig. 6b) 162 . Recently, the authors further integrated DART-seq with a single-cell RNA-sequencing platform and thus developed scDART-seq, achieving profiling of the m 6 A methylome in single cells 163 . In addition, some RNA exonuclease also possesses binding affinity for certain RNA modifications under certain conditions. For instance, in the presence of Ca 2+ , E. coli Endonuclease V (eEndoV) promotes binding of inosine in RNA instead of cleavage. Taking advantage of this approach, Endonuclease V immunoprecipitation enrichment sequencing (EndoVIPER-seq) can enrich A-to-I edited transcripts from cellular RNA 164 .
Similar to chemical-induced hydrolysis, some endonucleases also show distinct cleavage efficiency between modified and unmodified transcripts, which can be utilized to enrich modification-containing transcripts and identify modified nucleotides. For instance, the E. coli RNA endoribonuclease MazF can specifically cleave the unmethylated 5ʹ-ACA-3ʹ motif but not the 5ʹ-m 6 ACA-3ʹ motif 165 . Taking advantage of this specificity, RNAendoribonuclease-facilitated sequencing (m 6 A-REF-seq)/MAZTERseq allows quantitative profiling of m 6 A at single-nucleotide resolution (Fig. 6c) 166,167 . However, a major limitation of this approach is that it can only detect m 6 A in the ACA context, which is only a small portion (16-25%) of m 6 A-modified sites.

NANOPORE DIRECT RNA SEQUENCING-BASED DETECTION TECHNOLOGY
Next-generation sequencing-based detection technologies have been widely applied for transcriptome-wide RNA modification detection. However, limited by the sequencing length (from 50 to 300 bp) and distinct detection principles for different modifications, next-generation sequencing-based detection technologies cannot map diverse RNA modifications simultaneously. The development of the Oxford Nanopore Technologies (ONT) sequencing platform shows promise in overcoming these challenges. In contrast to next-generation RNA sequencing, nanopore sequencing can sequence RNA directly without the requirement of additional reverse transcription and PCR amplification, thus decreasing the biases caused by these steps [168][169][170][171] . Mechanistically, single-stranded RNA is driven through the nanopore by the motor protein and thus causes ionic current changes for a set of k nucleotides residing within the pore (kmer; typically, k is 5), which enables decoding of the nucleotide sequence by computational analysis (Fig. 7). In addition to sequence information, chemical modifications and secondary structure, which also influence RNA translocation in nanopores, can be determined directly by computational algorithms 172 . In addition, the reads generated by nanopore sequencing are long enough to cover the full length of a transcript, thereby enabling accurate identification of highly repetitive regions, spliced products and polyadenylation tail length.
To identify RNA modification from the nanopore RNA direction, robust computational algorithm analysis is vital. There are two major analysis strategies, including alterations of raw signal intensity analysis (signal intensity, dwell time and trace) and base-called "error" feature analysis (base quality, mismatch frequency, and deletion frequency) 170 . Furthermore, modifications can be identified by the algorithms previously trained with modified and unmodified kmer contexts or comparison with the nonmodified control samples. These computational algorithms have allowed the identification of several modifications, including m 6 A, m 7 G, m 5 C, hm 5 C, Ψ and Nm, using nanopore direct RNA sequencing 169,[172][173][174][175][176][177][178][179][180] . In addition, owing to the long sequencing length, the sequence information and modification landscape of SARS-CoV-2 can be determined simultaneously by nanopore direct RNA sequencing 181,182 . However, there are still many challenges and limitations for nanopore RNA sequencing-based detection technology. First, the sequencing error rate (~1-4%) 168 is still much higher than that of next-generation sequencing (~0.1-1%). Since some algorithms exploit systematic base-calling errors to identify RNA modifications, the high sequencing error rate increases the complexity for base calling and modification identification. Second, the throughput of nanopore RNA sequencing is relatively low (1-3 Gb per flow cell), and the costs of direct RNA sequencing are high. Recently, several studies have shown that accurate modification detection requires high sequencing coverage (at least 30X) 173,180 . Hence, the sequencing depth and costs limit broader application of this strategy. Third, nanopore direct RNA sequencing requires a large amount of RNA (~500 ng polyA+ RNA), and thus, it is not suitable for samples that are difficult to obtain. Therefore, efforts are still needed to improve nanopore RNA direct sequencing, including the development of more powerful nanopore proteins and robust computational algorithms.

DISCUSSION
Since all existing detection methods confer both advantages and drawbacks, appropriate detection strategies should be chosen by considering the abundance of RNA modifications, RNA types of interest (rRNA, tRNA, mRNA or miRNA), the amount of starting material, etc. For instance, direct sequencing methods preserve the modification status but are often confounded by other factors, including RNA secondary structure, SNPs, and sequencing errors. Direct sequencing methods also require a high sequencing depth to detect modifications in low-abundance RNA. Antibody-based detection technologies are powerful and capable of detecting lowabundance modifications in rare RNA species. However, due to the enrichment step, the stoichiometry information of modification is lost, and large starting materials are needed. In addition, since antibodies may have intrinsic bias on RNA sequences and secondary structures, caution is needed to eliminate false-positive signals. Chemical treatment-based approaches have been widely applied to RNA modification detection owing to their high labeling specificity. For this strategy, both high labeling efficiency and mild reaction conditions are required for transcriptome-wide modification detection and quantification. Metabolic labeling or RNA modificationrelated protein-based approaches provide an alternative strategy to characterize modified residues but can only be applied to cultured cells. Therefore, appropriate negative controls are necessary to reduce false positives, including modification status manipulation (enzyme knockdown/knockout), chemical or enzyme treatment, and differential RT conditions. Orthogonal locus-specific detection methods are also necessary to validate the modification sites and status. In addition, synthetic modified spike-in controls preserve stoichiometry information during sequencing 122,183 . In addition, synthetic RNAs can also be developed for reducing false positives in sequencing-based technologies. For instance, an elegant study, which utilizes an unmodified IVT RNA library resembling the endogenous transcriptome as a negative control, can precisely and quantitatively map m 6 A and m 5 C through calibration 184 .
Nanopore direct RNA sequencing has been applied to the detection of several RNA modifications, showing promise for the simultaneous identification of distinct modifications in a single molecule. However, the accuracy and sensitivity of nanopore direct RNA modification sequencing are limited, and the simultaneous detection of different modifications is still unachieved. Further efforts should be made to improve the instrument to reduce the signal noise and develop robust computational analysis to identify multiple modifications simultaneously. Finally, the development of RNA modification detection technologies for low-input and singlecell samples is still an urgent need for facilitating understanding of the roles of RNA modifications in physiological and pathological processes. Improvement in integrating existing RNA modification detection strategies with single-cell RNA sequencing platforms shows promise. Collectively, recent progress in detection technologies promotes functional studies of RNA modifications, and we anticipate that further advancement will lead to a more comprehensive understanding of the epitranscriptome.


Use of native chicken breeds (Gallus gallus domesticus) for the development of suitable methods of Cantabrian capercaillie (Tetrao urogallus cantabricus) semen cryopreservation

Abstract Background The Cantabrian capercaillie (Tetrao urogallus cantabricus) is critically endangered. This subspecies has the lowest genetic variability and it is in regression. It belongs to Phasianidae family; therefore, the domestic chicken (Gallus gallus domesticus) could be a good model for developing reproductive technologies for use in capercaillie populations with low availability of animals. Objectives In this study, we analyzed the response of capercaillie sperm to the freezing–thawing process for contributing to the development of a semen cryobank of Cantabrian capercaillie. Methods We used domestic chicken as the animal model in order to obtain the freezing protocol before applying on capercaillie. In the first experiment, two different extenders (EK and LR84) and different concentrations [4% and 6% dimethyl‐acetamide (DMA) v:v] of cryoprotectants were evaluated using in‐straw freezing method in domestic chickens. A pilot study in capercaillie males, using the same conditions evaluated in chicken, was performed. Results In chicken, we found that the LR84‐4% DMA media provided the best results for freezing semen. In capercaillie study, LR84 extender seemed to be the most appropriate diluent and 4% was the better dose of DMA cryoprotectant agent. Further, based on previous studies carried out in rooster samples, we also tested the glycerol (8% v/v) as a cryoprotectant for capercaillie semen cryopreservation. Conclusions Our results suggest that sperm from both domestic and wild species had a similar response to freezing–thawing processes. Mediterranean chickens may be used as a suitable model for developing sperm freezing protocols that can be extrapolated to threatened capercaillie populations. In addition, LR84 media with glycerol was the most efficient extender to freeze capercaillie sperm native.

cantabricus) inhabits deciduous forests of the Cantabrian Mountains of
Spain. This species faces a high risk of extinction in its native habitat, not only because of its peripheral location but also due to its small population size, low genetic diversity and low incoming gene flow (Alda et al., 2013). The primary causes of threat are multiple, such as the management of the habitat that involved increasing the density and transformation of forest edges, increasing predators, climate change and hunting. In 2019, the census carried out by the Ecological Transition Ministry of Spain (2019) reveals that there are only 292 specimens of Cantabrian capercaillie that survive in a 350 km 2 range of the ridge Cantabrian.
Conservation programmes and reproductive technologies can help to protect species from becoming extinct. Semen cryopreservation, coupled with artificial insemination, has become the main tool for threatened species in ex situ assisted reproduction. Also preserving their genetic diversity in genome resource banks is essential if the success of captive breeding programmes is to be guaranteed (Prieto et al., 2014).
Previous studies on the optimization of capercaillie semen cryopreservation protocols have been described in central European capercaillie populations (T. urogallus). Kowalczyk and Łukaszewicz (2015) reported two methods of freezing capercaillie semen using EK as extender and the permeant cryoprotectants 6% dimethyl-acetamide (DMA) for freezing in pellets and 6% of dimethyl-formamide (DMF) for freezing in straws. Their results showed that the straw method allows obtaining a higher survival rate and number of motile sperm, compared to pellets. However, there are no previous studies of cryoresistance of Cantabrian capercaillie semen. Reproductive techniques for use with a target species could be developed using phylogenetically related model species. Domestic chicken (Gallus gallus domesticus), a Phasianidae species as the capercalillie, might provide a good model for developing new reproductive technologies for use with threatened related wild species as capercaille, in which the number of available individuals is very scarce.
Based on the protocols developed in domestic chicken, we described, for the first time, the effect of different extenders and cryoprotectants in-straw freezing protocol of the Cantabrian capercaillie's semen. All semen collections in both chicken and capercaillie were performed using the dorsoabdominal massage technique (Burrows & Quinn, 1937;Łukaszewicz et al., 2011). In capercaillie males, semen was recovered by capillarity using a microhaematocrit tube (Brand GMBH + Co KG, Wertheim, Germany).

Assessment of sperm variables
The sperm concentration was estimated using a Neubauer chamber  equipped with a probe resistant to freezing. For thawing, the straws were warmed for 30 s in a water bath at 5 • C.

Experimental design
This study consisted of one experiment and a pilot study involving the cryopreservation of semen from native chicken breed and capercaillie, respectively. Experiment 1 involved the use of two different diluents with two different DMA concentrations in a Birchen Leonesa rooster.
The pilot study consisted of the use of capercaillie males, involving the same extenders with DMA as Experiment 1, and a Lake-Revie extender with glycerol as previously tested in native Mediterranean chicken breeds (Abouelezz et al., 2015).

Experiment 1: Cryopreservation of semen of the Birchen Leonesa rooster comparing two extenders and two different concentrations of DMA
This experiment studied the effect of two extenders, LR84 and EK, with two different concentrations of the DMA cryoprotectant (4% and 6%) on frozen-thawed rooster sperm variables.
Ten samples of five roosters (two for each animal) about 2 years old of the Birchen Leonesa were obtained. As the volume obtained from each male was not enough to perform the four treatments, half of the samples (n = 5) were immediately diluted 1:1 (v:v) using LR extender, and the other half of samples (n = 5) were diluted 1:1 (v:v) using EK extender. Then, each aliquot was refrigerated for 1 h at 5 • C. After the equilibrated period with the extender, a third volume of the extender with 12% DMA (final concentration 4% DMA) or 18% DMA (6% DMA final concentration) was added and equilibrated for 10 min at 5 • C.
After equilibration time, the samples were loaded into 0.25 ml French straws and frozen.
For this experiment, the comparison of each experimental condition was made analyzing the motility variables by CASA system, and the viability by fluorescence (PI/SYBR14) of the fresh and frozen/thawed rooster semen.

Pilot study: Cryopreservation of capercaillie semen comparing extenders previously studied in chicken
Under the hypothesis that the best conditions observed in roosters could be the same for capercaillie, we carried out this study using the same extenders and cryoprotectant agents previously evaluated in roosters.

2.8
Pilot study 2.1: Freezing semen of capercaillie using LR84 and EK extenders with 6% and 4% DMA as a cryoprotectant Semen samples of three capercaillie males were analyzed: one Cantabrian capercaillie male named URS1, and two European capercaillie males named URA1 and URA2. The samples obtained were divided into two aliquots. Both fractions were brought to a 1:1 (v/v) dilution, but each one with a different extender (EK or LR84), and refrigerated for 1 h at 5 • C. Then, DMA was added to leave a final 6% or 4% concentration and equilibrated for 10 min at 5 • C before freezing.
For this experiment, due to the scarce volume of the collected sample, the fresh sperm analysis consisted only the subjective motility and volume. For frozen/thawed samples, viability (PI/SYBR14), motility (CASA) and sperm DNA integrity (TUNEL) were analyzed. TA B L E 1 Rooster sperm quality variables (mean ± SE) of fresh and frozen-thawed samples with 4% of dimethyl-acetamide (DMA)

2.9
Pilot study 2.2: Freezing semen of capercaillie using LR84 extender and glycerol as a cryoprotectant The glycerol (GLY) as cryoprotectant was previously checked in native Mediterranean chicken samples (Abouelezz et al., 2015), returned higher sperm viability, intact acrosome and motility values. Thus, we tested the freezability of the Cantabrian capercaillie sperm with 8% GLY in the in-straw freezing method.
Samples from four of six Cantabrian capercaillie males (male identification: 31-C, 55-C, 61-C and 70-C) were obtained. Only the semen volume obtained from two of them was enough for freezing (31-C and 55-C). Both samples obtained were brought to a 1:1 (v/v) dilution in LR84 extender for 1 h at 5 • C. Then, the glycerol was added directly to diluted sperm samples to reach the final concentration of 8% (v/v).
For this experiment, the fresh sperm analysis consisting of the subjective motility, viability (eosin/nigrosin), sperm morphoabnormalities, acrosome status and DNA integrity were performed. For frozen/thawed samples, viability (PI/SYBR14 and E/N), motility and sperm DNA integrity (TUNEL) were analyzed.

Statistical analysis
The influence of the interaction semen media (LR84 and EK) × cryoprotectant concentration (DMA 4% and 6%) on sperm variables was analyzed by two-way analysis of variance (ANOVA), following the statistical model xijk = m + Ai + Bj + ABij + eijk, where xijk is the measured sperm variable, m is the overall mean of variable x, Ai is the effect of media (i = 1-2), Bj is the effect of DMA concentration (j = 1-2); ABij is the interaction between A and B and eijk is the residual (k = 1-10). A post hoc Newman-Keuls test was performed to compare differences between extenders. Comparisons between fresh and frozen-thawed sperm variables were made using a paired t test.
Data were expressed as means ± SE. All statistical calculations were made using Statistica software for Windows v.12 (StatSoft Inc., Tulsa, OK, USA).

RESULTS
In roosters, the values of all frozen/thawed sperm variables, except for motility score, linearity (LIN), straightness (STR) and wobble (WOB) were lower (p < 0.05) than those recorded for fresh sperm when the semen was cryopreserved using the 6% DMA (Table 1). In addition, the values of all frozen/thawed sperm variables except for LIN, WOB BCF were lower (p < 0.05) than those recorded for fresh sperm when the semen was cryopreserved using the 4% DMA (Table 1). Frozenthawed sperm viability, curvilinear velocity (VCL), straight-line velocity (VSL) and average path velocity (VAP) were greater (p < 0.05) using LR84-6%DMA extender than EK-6% DMA extender. There were no TA B L E 2 Capercaillie sperm quality variables of frozen-thawed samples using LR84 or EK extenders with 4% of dimethyl-acetamide (DMA) (Cantabrian capercaillie URS1) and 6% of DMA (European capercaillie URA1 and URA2) the percentage of total motility and progressive motility seems to be higher in samples frozen with LR84-4% DMA. However, the kinetic variables of motility (VCL, VSL, VAP, STR and ALH) were greater than using EK extender. In addition, the percentage of DNA fragmentation observed is higher in EK (4.5%) than LR84 extender (0%) ( Table 2, URS1 male).
The percentage of viable sperm after freezing was greater than those observed using DMA as a permeant cryoprotectant.

TA B L E 3
Cantabrian capercaillie sperm quality variables of frozen-thawed samples using LR84 extender with 8% of glycerol

DISCUSSION
In this study, we obtained and described the ejaculate of both  (Abouelezz et al., 2015), conversely with studies in red jungle fowl (Gallus gallus murghi) in which 6% DMA maintained higher postthaw quality and fertility (Rakha et al., 2017). Hence, it should be noted that the sperm of native Mediterranean breed of chicken are not very tolerant of DMA, and concentrations lower than 6% are recommended.
Despite suitable results have been found in semen European capercaillie frozen with 6% DMA (Kowalczyk et al., 2012), our results recommend that the lower DMA concentration should be used in future stud-ies with Cantabrian capercaillie. The method of freezing may also influence the results. The pellet method has been recommended when DMA is used as cryoprotectant (Tselutin et al., 1995). Indeed, first studies in capercaillie with DMA used the semen freezing in pellets (Kowalczyk et al., 2012). However, further studies by Kowalczyk and Łukaszewicz (2015) did not find differences between straw and pellet methods with 6% DMA as the cryoprotectant.
Previous findings (Abouelezz et al., 2015) showed that glycerol was more effective than DMA to cryopreserve sperm from native chicken breeds. The advantages of using glycerol as a cryoprotectant in chicken semen have also been established and recommend in the conservation of rare genetic resources (Thélie et al., 2019). In order to study if similar findings were found in capercaillie, we evaluated the effect of the  (Kowalczyk & Łukaszewicz, 2015). Our data showed that frozen-thawed sperm quality variables of the 31-C male were better than the 55-C male. In this regard, it is well known that the sperm ability to be cryopreserved can vary between individuals. Moreover, other earlier studies in European capercaillie indicated that the quality of ejaculates depends on individual male properties and their age but not the way of male management (Kowalczyk & Łukaszewicz, 2015).
The initial quality of fresh semen sample plays a relevant role in freezing-thawing suitability. The Cantabrian capercaillie volumes were lower in comparison with previous studies in European capercaillie (Ciereszko et al., 2011;Łukaszewicz et al., 2011). Also, sperm concentrations obtained in Cantabrian capercaillie were lower than those found in European capercaillie by Kowalczyk et al. (2012) and Kowalczyk and Łukaszewicz (2015). Perhaps, the volumes and concentration of the Cantabrian capercaillie ejaculated are naturally less than what is seen in European capercaillie located in central Europe, or maybe the bottleneck of the Cantabrian population determines a strong inbreeding that finally affects the semen quality.
The high DNA damage levels in fresh Cantabrian capercaillie semen samples of the pilot study 2.2 are remarkable (from 19% in 31-C to 28% in 55-C). Regarding this, a link has been reported between sperm DNA damage and inbreeding in ungulates (Ruiz-Lopez et al., 2010). Our data also showed that semen quality from European capercaillie males of the zoological garden also showed low quantitative characteristics, which may reveal inbreeding of this captive population.
Conversely, the quality of the fresh samples for Cantabrian capercaillie (URS1 male of the pilot study 2.1 and all males of the pilot study 2.2) was similar to those observed in European capercaillie in previous studies. While we observed 55%-80% of total motility in fresh semen, Kowalczyk and Łukaszewicz (2015) observed 82% average motility and Ciereszko et al. (2011) reported 71% motility after 2 h of short-term storage in EK extender. Fresh sperm viability, ranging between 77% and 85% in Cantabrian capercaillie, was similar to European capercaillie semen (Kowalczyk & Łukaszewicz, 2015;Kowalczyk et al., 2012;Łukaszewicz et al., 2011).

CONCLUSION
In conclusion, our results suggest that sperm from both domestic and wild species had a similar response to freezing-thawing processes.
Mediterranean chickens may be used as a suitable model for developing sperm freezing protocols that can be extrapolated to threatened capercaillie populations. In addition, LR84 media with glycerol was the most efficient extender to freeze capercaillie sperm native. Although the sample size of this study is small and it is necessary to carry out more studies to obtain consistent and comparable results, it should be noted that this is the first description of semen freezing in Cantabrian capercaillie for the future development of a germplasm bank in this subspecies.


s2fieldsofstudy: Biology Medicine

text:
E-cadherin deficiency promotes prostate macrophage inflammation and bladder overactivity in aged male mice

Decreased E-cadherin immunostaining is frequently observed in benign prostatic hyperplasia (BPH) and was recently correlated with increased inflammation in aging prostate. Homozygous E-cadherin deletion in the murine prostate results in prostate inflammation and bladder overactivity at 6 months of age. However, this model is limited in that while E-cadherin is significantly reduced in BPH, it is not completely lost; BPH is also strongly associated with advanced age and is infrequent in young men. Here, we examined the functional consequences of aging in male mice with prostate luminal epithelial cell-specific E-cadherin heterozygosity. In control mice, aging alone resulted in an increase in prostate inflammation and changes in bladder voiding function indicative of bladder underactivity. At 24 months of age, mice with prostate-specific Cre-mediated heterozygous deletion of E-cadherin induced at 7 weeks of age developed additional prostatic defects, particularly increased macrophage inflammation and stromal proliferation, and bladder overactivity compared to age-matched control mice, which are similar to BPH/LUTS in that the phenotype is slow-progressing and age-dependent. These findings suggest that decreased E-cadherin may promote macrophage inflammation and fibrosis in the prostate and subsequent bladder overactivity in aging men, promoting the development and progression of BPH/LUTS.

AGING BPH tissues [6], suggesting that aging could contribute to the increased prostatic inflammation and reduction in E-cadherin observed in BPH tissues.
Aging-related changes that are associated with BPH/LUTS have also been reported in mice, including an increase in prostatic inflammation and altered urinary function. Mice at 12 months of age have an increase in inflammatory infiltrates and increased vascularization compared to mice at 2 months of age [7,8]. At 24 months of age, the stromal compartment of murine prostate exhibits increased collagen deposition, disorganization of smooth muscle cells and extracellular matrix and an increase in inflammatory infiltrates -particularly T and B cells -compared to young animals [9]. Genes associated with extracellular matrix, cell adhesion and focal adhesion are downregulated in aged stromal and epithelial compartments in both the human and murine prostates [8,10]. Previous comparison studies of aged versus young mice revealed changes in bladder voiding function changes, including reduced bladder contractility and smaller voided volumes per micturition in both male and female aged mice [11,12]. Collectively, these studies suggest that similar to humans, the aging mouse undergoes lower urinary tract changes including prostatic stromal fibrosis and inflammation as well as changes in bladder function.
We recently generated a murine model with homozygous deletion of the E-cadherin gene in prostatic luminal epithelial cells to determine functional consequences of E-cadherin ablation in BPH pathogenesis [13]. While these mice developed prostatic inflammation and overactive bladder at 6 months of age [13], this does not match strong agingdependent development of LUTS in humans. This may be related to the findings that E-cadherin is only partially reduced in BPH luminal cells [14][15][16] and aging alone may induce changes in prostate and bladder [11,12]. Thus, to model the partial loss of E-cadherin expression on LUTS during aging in humans, we examined lower urinary tract function and pathology in aged transgenic mice with heterozygous deletion of E-cadherin in prostate luminal epithelial cells. Aged control mice displayed an increase in prostatic inflammation and changes in bladder voiding characteristics compared to control mice at 6 months of age, while aged mice with heterozygous deletion of E-cadherin displayed an increase in prostate macrophages, stromal proliferation and fibrosis, and bladder overactivity compared to age matched controls. Taken together, these findings suggest age-dependent changes induced by E-cadherin heterozygosity in the murine prostate could promote a BPH/LUTS-like phenotype in the prostate and bladder.

E-cadherin in human BPH and transgenic mouse prostate
BPH and LUTS are typically associated with aging, and seldom identified in patients under the age of 51 [2,3,17]. In a recent study of the impact of age on the lower urogenital tract in male mice, aging mice displayed prostatic enlargement and altered urinary function compared to young animals [11], illustrating the potential relevance of an aging rodent model to identify mechanisms driving BPH/LUTS in humans. Mice reach adulthood at approximately 10 weeks of age and undergo reproductive senescence from 15-18 months of age, while post 18 months of age is considered to be the post-reproductive senescence period for mice [18]. The correlate to human adulthood is 20-51 years of age, reproductive senescence from 51-78 years of age and post-reproductive senescence period over age 78 [18]. Since BPH/LUTS incidence increases from ~ 50% in men at the age of 50 and reaches approximately 80% in men over the age of 70 [17], we chose to examine the impact of E-cadherin loss in male mice at 24 months of age -corresponding to 75-85 years of age in humans ( Figure 1A). Previous studies examining the role of aging in the murine prostate have also examined mice at 24 months of age [9,11]. To examine the impact of aging, we compared mice at 24 months of age to young adult mice at 6 months of age -corresponding to ~30 years of age in humans ( Figure 1A). E-cadherin immunostaining of the epithelial layer in normal adjacent prostate to BPH and BPH glands was uniformly distributed and most intense along the cell membrane, with decreased staining observed in BPH glands as evidenced by a reduction of clearly demarked E-cadherin positive cell borders in BPH glands ( Figure  1B) [6]. This diffuse reduction in E-cadherin staining was not observed in mice with a homozygous deletion of the E-cadherin gene in luminal epithelial cells [13] ( Figure 1C). While these Cdh1 -/mice contain many prostate epithelial cells lacking E-cadherin staining, they are often found surrounded by E-cadherin positive luminal epithelial cells in a mosaic pattern [13] ( Figure  1C). This is likely the result of replenishment of luminal epithelial cells from a basal progenitor that was not ablated of the E-cadherin gene [19]. In order to more closely recapitulate the uniform reduction in E-cadherin expression observed in BPH glands of patients aged 50-77 years [6], we generated a cohort of mice with heterozygous deletion of E-cadherin and examined them at age 24 months. In this way, we could determine the impact of reduced E-cadherin expression (i.e., generated by heterozygosity in the mouse model) on the aged prostate and bladder. Similar to the pattern of AGING E-cadherin immunostaining observed in prostate specimens from BPH patients, mice at 24 months of age displayed cell membrane E-cadherin expression in the epithelial layer of both Control and Cdh1 +/mice, with a diffuse decreased expression in prostate glands of Cdh1 +/mice in all lobes of the prostate (blue arrows, Figure 1D, center panel, Supplementary Figure 1). Cdh1 +/mice also displayed glands with the mosaic Figure 1. Modeling the combined impact of heterozygous E-cadherin loss and aging on the murine prostate. (A) Correlation of human age and BPH/LUTS incidence to aging of C57BL/6 mice [18]. Experimental timepoints for mice at 6 months and 24 months of age (noted in bold) correlate to 30 years and 75-85 years of age for men. (B) Representative E-cadherin immunostaining (red) in normal prostate adjacent to BPH (left) and in glandular BPH (right) specimens from a 77-year-old patient. (C) E-cadherin immunostaining (brown) in Control (left) and mice with homozygous deletion of E-cadherin (Cdh1 -/-) (right) mouse ventral prostate at 6 months of age. E-cadherin immunostaining in Cdh1 -/mice displayed a mosaic pattern of E-cadherin negative cells (black arrows) surrounded by a layer of E-cadherin positive epithelial cells (blue arrows  Data represent mean ± Standard Deviation. pattern of E-cadherin negative cells surrounded by E-cadherin positive cells (black arrows, Figure 1D, right panel), similar to the pattern observed in Cdh1 -/mice at 6 months of age [13].

Impact of aging and E-cadherin deficiency on the murine prostate
Virgin male mice were generated with the following genotype combinations: PSA-Cre + ; Cdh1 flox/+ (referred to herein as Cdh1 +/-) and PSA-Cre -; Cdh1 flox/+ (Control). As an additional control for the possible effects of PSA-Cre, we also generated a cohort of three PSA-Cre + ; Cdh1 +/+ (PSA-Cre + ) mice for histologic analyses. At 24 months of age, no statistically significant differences were observed in mean body, organ, or prostate mass between Control and Cdh1 +/mice ( Table 1). The prostates from PSA-Cre + mice also appeared histologically normal and expressed Ecadherin in a pattern similar to PSA-Cre -; Cdh1 flox/+ controls (Supplementary Figure 2). There were no histologic defects detected in the kidney, spleen or testis and no difference in morbidity or mortality between groups at 24 months of age (data not shown). These findings suggest that the presence of PSA-Cre did not induce systemic defects.
At 6 months of age, the prostates of control and Cdh1 +/mice appeared histologically normal and did not display any epithelial pathologic changes ( Table 2). Aging in control mice induced a nonsignificant increase in prostate hyperplasia and mPIN (murine prostatic intraepithelial neoplasia). The prostates of Cdh1 +/mice at 24 months of age displayed a statistically significant increase in epithelial changes including epithelial hyperplasia and mPIN lesions compared to both agematched controls and Cdh1 +/mice at 6 months of age ( Table 2 Figure 2D, 2E). In a post hoc comparison of control mice at 24 months of age to mice at 6 months of age, there was a significant increase in both epithelial and stromal proliferation in aged mice ( Figure 3). We did not observe a significant increase in ECM deposition in aged mice compared to 6 months old controls (Supplementary Figure 4). These results suggest that aging is associated with an increase in epithelial and stromal proliferation, and that E-cadherin deficiency further increased stromal proliferation in the aged murine prostate.
We recently showed that in aging human prostate, increased inflammation is correlated with downregulation of epithelial barrier proteins E-cadherin [6], which was previously found to be down-regulated in BPH [14][15][16]. Previous studies have also demonstrated an association between macrophage infiltration and fibrosis in the murine prostate [20]. in the prostates of both controls (4/9) and Cdh1 +/mice (3/7) at 24 months of age, while neither cohort displayed lymphoma at 6 months of age (0/7 in controls, 0/3 in Cdh1 +/mice). Although the age-related increased incidence was not Table 2. Impact of age and E-cadherin deficiency on prostatic epithelial pathologic changes in male mice.

Voiding behavior and conscious cystometry in Cdh1 +/mice at 24 months of age
Previously, we and others have reported an increased urinary frequency [22] and bladder overactivity in mice and rat models of bacterial-or chemical-induced prostatic inflammation [23,24]. We also reported that mice with homozygous deletion of Cdh1 developed prostatic inflammation and displayed an increase in the number of voids, non-voiding contractions and a decrease in the average single voided volume compared to controls at 6 months of age [13]. Post-hoc comparison of control mice at 6 months of age vs. 24 months of age revealed that aged mice displayed a significant decreased average single voided volume in voiding assays during the four-hour observation period despite larger bladder mass at necropsy and a trend increase of ICI during cystometry in 24 months-old control mice (Table 3). Because bladder voiding efficiency calculated as a percent ratio of voided volume against bladder capacity was lowered to 66% in aged control mice compared to control mice at 6 months of age (Table 3), it is assumed that aged mice exhibited an underactive bladder condition resulting in inefficient voiding, in line with a previous study in which voiding dysfunction due to reduced bladder contractility was observed with aging in male mice [12]. In addition, there was a trend decrease in total AGING urine output volume (1101.2 vs. 567.7, p = 0.07) during four hours in control mice at 24 months of age compared to control mice at 6 months of age (Table 3).
Although the difference was not statistically significant, the measured bladder volume at necropsy of Cdh1 +/mice at 24 months of age was much larger than that of age-matched control mice (3.31 vs. 0.931 cm 3 , p = 0.068) ( Table 3). In accordance, aged Cdh1 +/mice displayed trend increases in ICI (p = 0.090) and bladder capacity (p = 0.053) in cystometry compared to agematched controls (Table 3) although enlarged bladders did not display obvious histological defects compared to age-matched controls ( Figure 6). However, because bladder voiding efficiency was not different between aged Cdh1 +/mice and age-matched control mice (56 vs. 66%, p = 0.478) (Table 3), the aging-related underactive bladder condition does not seem to be significantly worsened by E-cadherin downregulation. In contrast, cystometric tracing of awake Cdh1 +/mice demonstrated a significantly increased mean number of non-voiding contractions (NVC) per minute compared to controls (Figure 7) in aged mice, suggesting that prostatic defects induced by E-cadherin heterozygosity contributed to the development of bladder overactivity during the storage phase in aged mice. Cystometric analysis was not performed on Cdh1 +/mice at 6 months of age since they did not display any prostatic defects. Taken together, these changes suggest that aging contributes to bladder underactivity during the voiding phase function while decreased E-cadherin in the aging prostate can additionally induce bladder overactivity during the storage phase in aged mice.

DISCUSSION
We previously demonstrated that homozygous Cdh1 knockout in prostate luminal epithelial cells induced prostatic inflammation and hyperplasia and subsequent bladder overactivity in mice at 6 months of age [13].
Thus, E-cadherin plays an important role in maintenance of adult prostate and prostate inflammation induced by E-cadherin deficiency can cause alterations in bladder function. However, in benign prostatic conditions (i.e., BPH), E-cadherin is reduced but not completely ablated. Here, we sought to determine whether partial loss of E-cadherin rather than complete ablation could also contribute to the development of a At 6 months of age, neither control nor Cdh1 +/mice displayed any histologic defects in the prostate, but mice in both groups developed a significant increase in hyperplasia and mPIN lesions at 24 months of age, suggesting that aging contributes to prostate epithelial proliferation. Cdh1 +/mice displayed an increase in hyperplastic and neoplastic lesions compared to agematched controls ( Table 2). The increased incidence of both hyperplastic and neoplastic lesions in aged Cdh1 +/mice is not surprising. Both BPH/LUTS and prostate cancer are strongly associated with age and with decreased E-cadherin expression, however they are two distinct diseases [6,14,16,25,26]. Although human BPH is not thought to become prostate cancer and is not associated with an increase in prostate cancer risk, over 80% of prostate cancer patients also have histological BPH [27]. Furthermore, prostate cancer, at least in early stages, is not associated with LUTS [28]. Thus, it may not be surprising that we observe age-dependent effects in Cdh1 +/mice that extend beyond benign alterations, specifically, an enhancement in the incidence rate of mPIN (44% in control vs. 100% in Cdh1 +/-, p = 0.0337 in ventral prostate). These results suggest that prostate cancer in aged men may not only be associated with complete loss of E-cadherin expression [26], but also be promoted by even subtle changes in cellular function requiring optimal functioning of E-cadherin.   The development of LUTS in BPH patients is associated with elevated prostate inflammation [25]. At 6 months of age, homozygous deletion of E-cadherin in the murine prostate induced an increased number of non-voiding contractions, an increased number of voids and a smaller average voiding volume without changes in total urine volume [13]. However, bladder dysfunction in mice may not require a complete ablation of E-cadherin and be driven by aging. Aged control mice also displayed increased prostate inflammation ( Figure 5) and reduced voided volume compared to controls at 6 months of age ( Table 3). The average bladder mass of aged control mice was about twice that of controls at 6 months of age; and although the differences were not statistically significant, an associated trend increase in bladder capacity (i.e., increased ICI) with small voided-volume per micturition and reduced bladder voiding efficiency in aged compared to young animals suggest that aging mice develop an underactive bladder condition (Table 3). In aged Cdh1 +/mice, the average bladder volume was about three times that of age-matched controls at necropsy, and bladder capacity in cystometry tended to be increased (Figure 7, Table 3). However, bladder voiding efficiency was not significantly different between aged Cdh1 +/mice and age-matched controls ( Table 3), suggesting that the aging-related underactive bladder condition during the voiding phase was not significantly worsened by E-cadherin heterozygosity. In contrast, aged Cdh1 +/mice displayed a significant increase in the number of nonvoiding contractions during the storage phase ( Figure 7, Table 3), indicative of an overactive bladder condition. Taken together, these findings suggest that E-cadherin deficiency associated with increased prostatic inflammation in the aging prostate can contribute to the development of bladder overactivity as evidenced by increased NVC during the storage phase in aged mice, possibly due in part to prostatic macrophage infiltration and fibrosis.
Chronic low-grade systemic inflammation is a hallmark of aging (termed inflammaging) [1] and plays a role in the pathogenesis of several age-related chronic diseases [2]. Accumulating effects from redox stress, mitochondrial damage, senescence, epigenetic modifications, inflammatory disease, and frailty likely all play a role in promoting inflammaging (Reviewed in [3]). BPH is a prevalent prostate disease that has been strongly correlated with increased age and is frequently associated with inflammation [4]. Aging contributes to an increase in prostatic inflammation, and is a significant risk factor for the development and progression of BPH/LUTS [29]. It has been shown that aged mice in both sexes similarly develop characteristics of voiding dysfunction with low bladder voiding efficiency due to reduced bladder muscle contractility, which mimics the condition of underactive bladder often observed in aged humans [12]. Furthermore, although cell proliferation in the rodent prostate cannot directly compress the urethra due to a lack of capsular structure enclosing the prostate, unlike humans, aged male mice reportedly develop bladder outlet obstruction due to increased collagen deposition and glandular hyperplasia within the prostatic urethra [11,30]. Thus, both bladder underactivity and partial urethral obstruction are likely to contribute to voiding dysfunction detected in aged male mice with or without E-cadherin heterozygosity in this study. In addition, we recently showed that decreased E-cadherin in the human prostate was associated with aging and was correlated with increased inflammation [6], and we and others have shown that E-cadherin is decreased in BPH [14][15][16]. This study demonstrated that E-cadherin downregulation with enhanced prostatic inflammation further promoted BPH-like phenotypic changes in the prostate and bladder overactivity, which is one of the hallmark manifestations of male LUTS associated with BPH, in addition to voiding dysfunction, to which the aging process seems to be an important contributing factor. Taken together, our findings here demonstrate that aging promotes the prostate inflammation and changes in bladder function in Cdh1 +/mice which are characteristic of age-related diseases such as BPH/ LUTS.
Overall, these results demonstrate that age-dependent prostatic defects specific to Cdh1 +/mice include increased macrophage recruitment, an increased incidence in prostate stromal hyperplasia and extracellular matrix deposition as well as bladder overactivity. Aging contributes to an increase in prostate inflammation evident as increased presence of T-cell, B-cell, and macrophages, and declines in voiding function evident as large bladder capacity and decreased voided volume per micturition with low bladder voiding efficiency. The presence of macrophages in the prostate can be further exacerbated by E-cadherin loss potentially contributing to an increase in proliferation and stromal fibrosis, additionally inducing bladder overactivity evident as increased NVCs in aged mice. However, urinary frequency in aged mice was not significantly affected by E-cadherin heterozygosity, possibly due to an aging-induced reduction in bladder sensation. Heterozygous E-cadherin deficiency in the prostate of aged mice induced a milder phenotype than that previously reported mice with homozygous E-cadherin deletion [13], providing a tool to study the impact of more mild to moderate, chronic E-cadherin deficiency in aged mice. This slow progressing, agedependent phenotype induced by prostate-specific heterozygous E-cadherin loss recapitulates key characteristics observed in human BPH and LUTS and is distinct from other rodent models of BPH/LUTS. This model will be a useful resource for exploring whether targeting prostate inflammation or more specifically, macrophage inflammation or fibrosis, via pharmacologic or lifestyle interventions can delay or reduce the development or progression of symptomatic BPH/LUTS.

E-cadherin immunostaining in human prostate specimens
E-cadherin immunostaining was performed on sections from a previously described experimental cohort of prostate tissue specimens [6,31], which included 14 patients who received simple prostatectomy for symptomatic BPH, and six young healthy organ donors without any history of chemo-, radio-, or hormone therapy [31]. BPH specimens were composed of mixed hyperplastic nodules with both glandular and stromal expansion. The "deidentified" specimens were retrieved from the clinical files of UPMC by the University of Pittsburgh Biospecimen Core (PBC) with approval from the University of Pittsburgh Institutional Review Board for this research project under protocol #17010177. PBC also provided deidentified pathology reports for the patients constituting the study cohort through their IRB-approved Honest Broker System, HB015, to ensure research integrity. All participating patients or their next of kin provided informed consent for the banking protocol.
Samples were fixed in 10% formalin for at least 24 hours, then embedded in paraffin, sectioned at 5 μm.
Tamoxifen induction of Cre activity in male mice containing the PSA-CreER T2 allele was performed as previously for 5 consecutive days at 7 weeks of age [13,34]. Tamoxifen (Sigma Chemical Co., St. Louis, MO, USA) suspended in Kolliphor EL (Sigma) 3 mg/40 g of body weight was injected i.p. daily. All animals in the study were treated with the same regimens of tamoxifen injections and were examined at 6 months or 24 months of age. Body mass was measured using a triple beam balance immediately prior to euthanization. Length was determined by measuring the animal from the nose to the anus [35]. Bladder was measured in situ in three dimensions with a precision caliper as described [36] and bladder volume was estimated by calculating the volume of an ellipsoid in cubic millimeters [37]. The heart, spleen, left kidney, left testis and lower urinary tract were removed and fixed in 10% formalin. Fixed tissues were cleaned of excess fat and membrane with phosphate-buffered saline; mass of each organ and each prostate lobe was determined after blotting with filtration paper to remove excess liquid.

Histopathologic analysis of murine specimens
Murine tissue samples were fixed in 10% formalin for at least 24 hours, then embedded in paraffin, sectioned at 5 μm, and stained with hematoxylin and eosin. All tissues were examined by a board-certified animal pathologist in a blinded fashion (LHR, V.M.D.). Prostatic defects were identified as epithelial hyperplasia, stromal hyperplasia, inflammation and mPIN and adenocarcinoma per the criteria published by Shappell, et al., commonly used to score prostate lesions in transgenic mouse models [38].

Immunohistochemistry and Masson's trichrome staining of murine specimens
Immunohistochemical stains were performed on fivemicron sections of paraffin-embedded murine tissue specimens as described previously [39]. Briefly, sections were de-paraffinized and rehydrated through a graded series of ethanol. Heat-induced epitope retrieval was performed using a BioCare Decloaking Chamber ( Mean Ki-67-positive cell density was determined by analysis of sections from at least six independent mice from each genotype and from all lobes of the prostate. The average number of Ki-67-positive luminal epithelial cells or stromal cells in each prostate lobe for each mouse was determined from at least four nonoverlapping fields imaged at 40× magnification. The average number of CD19-positive B cells, CD68positive macrophages and CD3-positive T cells was also determined similarly for each prostate lobe in each mouse. Extracellular matrix (ECM) was quantified by calculating the average intensity of blue staining in Masson's trichrome stain using Adobe PhotoShop 2021, as described [40]. Seven fields from each section were analyzed and an average score was determined for each mouse.

Void spot assay
Mice were placed in metabolic cages and voiding behavior was evaluated for 4 hours. Filter paper was placed under the wire mesh bottom of each metabolic cage and urine stains on the paper were analyzed to determine voiding behavior and voided volumes using the Voided Stain on Paper (VSOP) method [41]. Before each analysis, mice were treated with subcutaneous injections of sterile water (20 µl/g body weight) to enhance urine production. The voided volume for each urine spot was calculated using ImageJ (NIH, Bethesda, MD, USA).

Cystometry
Mice were examined by cystometric investigation under a conscious condition as previously [23]. Under isoflurane anesthesia, following a lower midline abdominal incision, the bladder was exposed and catheterized with PE-50 tubing (Clay Adams Division of Becton Dickinson, Parsippany, NJ, USA) inserted through the bladder dome. After recovery from anesthesia, conscious mice were placed in a restraining cage (Economy holder 15 to 30 g, Kent Scientific, Torrington, CT, USA). After 1-2 hours of acclimation, saline was infused into the bladder at 0.01 ml/min to induce micturition. At least three reproducible micturition cycles were recorded after an initial stabilization period (60 min). After several micturition cycles, the saline infusion was stopped to evaluate intercontraction intervals (ICI), and non-voiding contractions (NVCs), bladder capacity and bladder voiding efficiency using Chart software (AD Instruments, Colorado Springs, CO, USA). NVCs were defined as rises in intravesical pressure that exceeded 20% of voiding pressure over the baseline without fluid elimination from the urethral orifice during bladder filling.
Post-hoc comparison of data from control mice at 24 months of age (PSA-Cre-; Cdh1 flox/+ ) to previously generated control mice at 6 months of age (PSA-Cre-; Cdh1 flox/flox ) [13] was performed to determine whether prostate inflammation and bladder function were altered with aging. Control mice from both 6 months and 24 months of age cohorts were treated with tamoxifen at 7 weeks of age.

Statistical analysis
Comparisons between groups were calculated using the student's t-test or two-tailed Fisher exact test using the method of summing small p-values as appropriate. The Pearson correlation coefficient was used to determine the correlation between extracellular matrix density, proliferation, and infiltration of inflammatory cells in the combined samples from aged mouse prostate (control and Cdh1 +/-). A p-value of p < 0.05 was considered significant. GraphPad Prism version 9 was used for graphics (GraphPad Software, San Diego, CA, USA). Values are expressed as mean ± S.D.

AUTHOR CONTRIBUTIONS
LEP and ZW designed experiments. LEP, TI, SM, WC, and CGM performed research experiments and analyzed and interpreted data. LHR analyzed murine prostate pathology and RD analyzed human prostate pathology. LEP wrote the manuscript with assistance from CGM, WB, DBD, NY and ZW. All authors reviewed and edited the manuscript.